ABSTRACT What are the thermodynamic driving forces that influence the free energy of membrane protein folding and assembly in lipid bilayers? For soluble proteins, the burial of hydrophobic groups away from aqueous interfaces is a major driving force, but membrane-embedded proteins cannot experience hydrophobic forces, as the lipid bilayer lacks water. A fundamental conundrum thus arises: how does a greasy protein surface find its greasy protein partner in the greasy lipid bilayer to fold faithfully into its native structure? Recently, a structurally stable and functional monomeric form of the normally homodimeric Cl-/H+ antiporter CLC-ec1 was designed by introducing tryptophan mutations at the dimer interface. We have used this to develop a new model system for studying reversible dimerization in membranes for free energy measurements, which simplifies the protein folding process while still encompassing all of the thermodynamic properties of protein interactions in the membrane environment. To quantify monomer vs. dimer populations across a wide range of protein per lipid mole ratios, we developed (i) Frster resonance energy transfer (FRET) and (ii) single- molecule photo bleaching by total internal reflection microscopy in liposomes methods for the CLC-ec1 system. The sensitivity of single-molecule microscopy allows us to go to extremely dilute conditions where we observe dissociation of CLC-ec1 in membranes. With measurements of the energetics already in place, we will investigate two alternative hypotheses that have pervaded discourse in this field. First, that protein association is enthalpy-driven by van der Waals forces at highly complementary surfaces. Changes in free energy will be measured upon substitution of interface residues to alanine or tryptophan, and efforts made to identify if VDW motifs can be conferred to already destabilized constructs. The second hypothesis is that interactions are driven by increased entropy of lipids upon subunit association. To study this, the molecules forming the lipid solvent will be modified by testing hydrophobic mismatch as a function of acyl chain length, and also the depletion-attraction force by changing lipid radius of gyration, e.g. larger unsaturated and tetraether lipids vs. smaller non-polar general anesthetics. For all experiments, free energy relationships will be measured as a function of temperature to extrapolate enthalpy and entropy changes. This research will be carried out by a team of interdisciplinary scientists in the Robertson laboratory, with levels of training from graduate student, postdoc, research scientist and principal investigator, combining expertise of membrane protein biochemistry, single-molecule microscopy and computational modeling to provide an unlimited investigation into this important biophysical question. The results from this study will provide a physical foundation for the development of informed strategies aimed at correcting protein mis-folding or regulating protein interactions in membranes in physiologically and pathological situations.